Excess NO generation plays a major role in the hypotension and systemic vasodilatation characteristic of sepsis. Yet the kidney response to sepsis is characterized by vasoconstriction resulting in renal dysfunction. We have examined the roles of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the renal effects of lipopolysaccharide administration by comparing the effects of specific iNOS inhibition, L -N 6 -(1-iminoethyl)lysine (L-NIL), and 2,4-diamino-6-hydroxy-pyrimidine vs. nonspecific NOS inhibitors (nitro-L -arginine-methylester). cGMP responses to carbamylcholine (CCh) (stimulated, basal) and sodium nitroprusside in isolated glomeruli were used as indices of eNOS and guanylate cyclase (GC) activity, respectively. LPS significantly decreased blood pressure and GFR (112 Ϯ 4 vs. 83 Ϯ 4 mmHg; 2.66 Ϯ 0.29 vs. 0.96 Ϯ 0.22 ml/min, P Ͻ 0.05) and inhibited the cGMP response to CCh. GC activity was reciprocally increased. L-NIL and 2,4-diamino-6-hydroxy-pyrimidine administration prevented the decrease in GFR (2.71 Ϯ 0.28 and 3.16 Ϯ 0.18 ml/min, respectively), restored the normal response to CCh, and GC activity was normalized. In vitro application of L-NIL also restored CCh responses in LPS glomeruli. Neuronal NOS inhibitors verified that CCh responses reflected eNOS activity. L-NAME, a nonspecific inhibitor, worsened GFR (0.41 Ϯ 0.15 ml/min), a reduction that was functional and not related to glomerular thrombosis, and eliminated the CCh response. No differences were observed in eNOS mRNA expression among the experimental groups. Selective iNOS inhibition prevents reductions in GFR, whereas nonselective inhibition of NOS further decreases GFR. These findings suggest that the decrease in GFR after LPS is due to local inhibition of eNOS by iNOS, possibly via NO autoinhibition. ( J. Clin. Invest. 1997. 100:439-448.)

Abstract-Asymmetric dimethylarginine (ADMA), which inhibits NO synthase, is inactivated by N G ,N G -dimethylarginine dimethylaminohydrolase (DDAH). We tested whether DDAH-1 or -2 regulates serum ADMA (S ADMA ) and/or endothelium-derived relaxing factor (EDRF)/NO. Small inhibitory (si)RNAs targeting DDAH-1 or -2, or an siRNA control were given intravenously to rats. After 72 hours, EDRF/NO was assessed from acetylcholine-induced, NO synthase-dependent relaxation and 4-amino-5-methylamino-2Ј,7Ј-diflouroflourescein diacetate for NO activity in isolated mesenteric resistance vessels (MRVs). Expression of mRNA for DDAH-1 versus -2 was 2-and 7-fold higher in the kidney cortex and liver, respectively, whereas expression of DDAH-2 versus -1 was 5-fold higher in MRVs. The proteins and mRNAs for DDAH-1 or -2 were reduced selectively by 35% to 85% in the kidney cortex, liver, and MRVs 72 hours following the corresponding siRNA. S ADMA was increased only after siDDAH-1 (266Ϯ25 versus; PϽ0.005), whereas EDRF/NO responses and NO activity were not changed consistently by siDDAH-1 but were greatly reduced after siDDAH-2. Mean arterial pressure was not changed significantly by any siRNA. In conclusion, S ADMA is regulated by DDAH-1, which is expressed at sites of ADMA metabolism in the kidney cortex and liver, whereas EDRF/NO is regulated primarily by DDAH-2, which is expressed strongly in blood vessels. This implies specific functions of DDAH isoforms. Key Words: RNA interference Ⅲ hypertension Ⅲ kidney Ⅲ blood vessel Ⅲ endothelium T he endothelium dependent relaxing factor (EDRF) response of resistance vessels is mediated predominantly by NO and an endothelium-dependent hyperpolarizing factor (EDHF). 1,2 Defects in NO occur in blood vessels and the kidneys of hypertensive models, despite often well-preserved expression of constitutive NO synthase (NOS). 3 One candidate to account for this paradox is superoxide (O 2 . ), which can inactivate NO in blood vessels. 4 A second candidate is asymmetric dimethylarginine (ADMA), which inhibits NOS activity, EDRF/NO responses, and L-arginine transport into cells by system y ϩ . 5 Arginine moieties in proteins are methylated by protein arginine methyltransferases. 5 Following protein catabolism, ADMA or its stereoisomer, symmetric dimethylarginine (SDMA), are released within cells and exported into the plasma. SDMA does not inhibit NOS. 6 Many of the patient groups or animal models at risk for cardiovascular disease have endothelial dysfunction and elevated serum levels of ADMA (S ADMA ). 5,7 Although S ADMA is a strong predictor of future cardiovascular events in high-risk patients, 8 it is presently unclear whether these associations are causative.ADMA and L-monomethyl arginine are metabolically inactivated by DDAH, whereas SDMA is not a substrate for this enzyme. 5 DDAH is expressed extensively in the proximal tubules of the rat kidney and the liver. 9,10 However, DDAH is expressed as 2 isoforms in rats and humans. 9 Current studies have shown that a 50% gene deletion for DDAH-1 i...

Abstract-The spontaneously hypertensive rat (SHR) exhibits angiotensin II (Ang II)-dependent oxidative stress and reduced efficiency of renal oxygen usage (Q O2 ) for tubular sodium transport (T Na ). We tested the hypothesis that oxidative stress determines the reduced T Na :Q O2 ratio in the clipped kidney of the early 2-kidney, Key Words: hypertension, renovascular Ⅲ Goldblatt hypertension Ⅲ renal artery Ⅲ nitric oxide R enal oxygen consumption (Q O2 ) is closely related to the energy required for sodium transport (T Na ). Classic studies have established that variations in sodium delivery and hence, transport, over a broad range are matched by proportionate changes in Q O2.1,2 Prevention of glomerular filtration reduced Q O2 to a low but measurable value, identified as the O 2 required for basal kidney metabolism. However, across a broad range of glomerular filtration rates (GFRs), Q O2 rose linearly with the GFR above the basal level. This defines the normal rate at which O 2 is consumed to satisfy the energy requirements for T Na (15 to 25 mol of Na transported per mol of O 2 consumed).Recent studies have reported that the T Na :Q O2 ratio is variable. Laycock and associates 3 showed that the T Na :Q O2 in the dog kidney was reduced by Ϸ50% during inhibition of nitric oxide (NO) synthase with L-nitroarginine. We showed a similar reduction in T Na :Q O2 in kidneys from spontaneously hypertensive rats (SHR). 4 The SHR is a model of reduced renal NO bioactivity associated with increased superoxide radical (O 2 ⅐Ϫ). 5 There is a complex interrelation between NO, O 2 ⅐Ϫ, and PO 2 or O 2 usage in the tissues. In pulmonary arteries and vascular smooth muscle cells, both chronic hypoxia and hyperoxia can enhance O 2 ⅐Ϫ levels. 6,7 Increased O 2 ⅐Ϫ interacts with NO, which reduces its bioactivity and produces peroxynitrite. Nevertheless, we detected a reduced renal cortical pO 2 in the SHR, which we attributed to inefficient utilization of O 2 for T Na as a consequence of functional NO deficiency during oxidative stress. We found also that the renal cortical hypoxia and reduction in PO 2 in the SHR could be corrected by prolonged administration of the angiotensin receptor antagonist candesartan (Cand) but not by equally effective antihypertensive therapy with agents that do not block the renin-angiotensin system. Moreover, we found that Cand also restored NO bioactivity in the SHR kidney. 8 Angiotensin II (Ang II) stimulates the expression of NADPH oxidase. 9,10 We concluded that the reduced NO bioactivity and inefficient utilization of O 2 in the SHR kidney could have been secondary to oxidative stress. The present study was designed to test this hypothesis in the early phase of 2-kidney, 1-clip (2K,1C) Goldblatt hypertension. The 2K,1C is a pathophysiological model of Ang II-dependent hypertension, which suppresses function in the clipped kidney in response

Abstract-The angiotensin II (Ang II) slow-pressor response entails an increase in mean arterial pressure and reactive oxygen species. We used double-stranded interfering RNAs (siRNAs) in Sprague Dawley rats in vivo to test the hypothesis that an increase in the p22 phox component of NADPH oxidase is required for this response. Reactive oxygen species were assessed from excretion of 8-isoprostane prostaglandin F 2␣ and blood pressure by telemetry. Two siRNA sequences to p22 phox (sip22 phox ) reduced mRNA Ͼ85% in cultured vascular smooth muscle cells. Rats received rapid (10 second) IV injections (50 to 100 g) Key Words: hypertension, arterial Ⅲ arterioles Ⅲ oxidative stress Ⅲ kidney A ngiotensin II (Ang II) has been assigned a critical role in the generation and complications of human essential hypertension, yet plasma renin activity and plasma concentrations of Ang II are not remarkably elevated. 1 Mice, 2 rats, 3,4 rabbits, 5 or humans 6 infused with Ang II at doses that are initially subpressor develop a "slow-pressor response" in which the blood pressure (BP) increases progressively despite plasma Ang II concentrations that are increased only moderately. 3 The kidney is implicated in the slow-pressor response, because the development of hypertension depends on salt intake. 3 Moreover, rats or rabbits infused with Ang II have enhanced renal vasoconstriction to Ang II 5,7 despite downregulation of Ang II type 1 receptors.Reactive oxygen species (ROS) and superoxide anion (O 2 ⅐Ϫ ) have been implicated in the development of hypertension in the Ang II slow-pressor model, because hypertension is prevented by antioxidant molecules, such as a permeabilized form of superoxide dismutase or tempol, which is an superoxide dismutase mimetic nitroxide. 2,4,5,8 Infusions of Ang II increase the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in blood vessels 9 and the kidney cortex. 4,10,11 This complex enzyme, which was first described in phagocytes and, later, in blood vessels and the kidney, is composed of membrane-associated components of the flavoprotein catalytic core, gp91phox (now named Nox-2) and p22phox . 12 Activation requires phosphorylation of p47 phox 13and its assembly with p67 phox 14 and Rac-1 at the membrane. 12 Homologues of Nox-2 include Nox-1, which has been characterized in vascular smooth muscle cells (VSMCs), 9 and Nox-4, which has been characterized in the kidney. 15 VSMCs and kidneys have the phagocytic NADPH oxidase components. 12 p22 phox is expressed in the thick ascending limb, macula densa, distal convoluted tubule, collecting ducts, vasculature, and interstitial fibroblasts of the kidney. 16 It is believed to dock the enzyme complex in the cell membrane and stabilize Nox proteins. 12 There is colocalization of p22 phox and O 2 ⅐Ϫ generation in atherosclerotic plaques from human blood vessels. 17 The p22 phox component is upregulated in the

Tempol corrects hypertension without a compensatory sympathoadrenal activation or salt retention. The response is independent of nitric oxide, endothelin, or catecholamines and occurs despite increased PRA. It is accompanied by a reduction in oxidative stress and is maintained during increased salt intake.

Tempol is an amphipathic radical nitroxide (N) that acutely reduces blood pressure (BP) and heart rate (HR) in the spontaneously hypertensive rat (SHR). We investigated the hypothesis that the response to nitroxides is determined by SOD mimetic activity or lipophilicity. Groups (n ϭ 6 -10) of anesthetized SHRs received graded intravenous doses of Ns: tempol (T), 4-amino-tempo (AT), 4-oxotempo (OT), 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-1), 3-carbamoyl-proxyl (3-CP), or 3-carboxyproxyl (3-CTPY). Others received native or liposomal (L) Cu/Zn SOD. T and OT are uncharged, AT is positively charged and cell-permeable, and CAT-1 is positively charged and cell-impermeable. 3-CP and 3-CTPY have five-member pyrrolidine rings, whereas T, AT, OT, and CAT-1 have six-member piperidine rings. T and AT reduced mean arterial pressure (MAP) similarly (Ϫ48 Ϯ 2 mmHg and Ϫ55 Ϯ 8 mmHg) but more (P Ͻ 0.05) than OT and CAT-1. 3-CP and 3-CTPY were ineffective. The group mean change in MAP with piperidine Ns correlated with SOD activity (r ϭ Ϫ0.94), whereas their ED50 correlated with lipophilicity (r ϭ 0.89). SOD and L-SOD did not lower BP acutely but reduced it after 90 min (Ϫ32 Ϯ 5 and Ϫ31 Ϯ 6 mmHg; P Ͻ 0.05 vs. vehicle). Pyrrolidine nitroxides are ineffective antihypertensive agents. The antihypertensive response to piperidine Ns is predicted by SOD mimetic action, and the sensitivity of response is by hydrophilicity. SOD exerts a delayed hypotensive action that is not enhanced by liposome encapsulation, suggesting it must diffuse to an extravascular site. (39), by enhancing the peripheral sympathetic nervous system (37, 38), or by enhancing renal tubular NaCl reabsorption (17,22,23).Oxidative stress accompanies hypertension in many models of hypertension, including the spontaneously hypertensive rat (SHR) (25). Mitchell, Krishna, and colleagues (15,18,24) have shown that tempol (T) is a permeant amphipathic radical nitroxide (N) that detoxifies oxygen metabolites by redox cycling through one-electron transfer reactions. The nitroxide/ oxoammonium cation pair form an efficient redox coupling that mimics the enzymic action of SOD and confers catalaselike action to heme proteins (14, 15). Although T lowers blood pressure (BP) in many animal models of hypertension accompanied by oxidative stress, including the SHR (9, 21, 26, 27, 29, 36 -38), the mechanisms of its in vivo action are not clearly established.Fink and colleagues (38) have shown that T given to deoxycorticosterone acetate-salt rats reduces BP before it has dissipated O 2 Ϫ ⅐ in the aorta. This acute antihypertensive response is accompanied by reduced renal sympathetic nervous system activity. It is unclear whether these neural actions of T depend on SOD mimetic action. Nevertheless, intravenous injection of liposomal (L), polyethylene-glycol, or heparin-bonded SOD lowers BP in SHR (20) or ANG-II-infused hypertensive rats (19) or restores ACh-induced relaxation in blood vessels from atherosclerotic rabbits (34).We investigated the hypothesis t...

Acute intravenous Tempol reduces mean arterial pressure (MAP) and heart rate (HR) in spontaneously hypertensive rats. We investigated the hypothesis that the antihypertensive action depends on generation of hydrogen peroxide, activation of heme oxygenase, glutathione peroxidase or potassium conductances, nitric oxide synthase, and/or the peripheral or central sympathetic nervous systems (SNSs). Tempol caused dose-dependent reductions in MAP and HR (at 174 micromol/kg; DeltaMAP, -57+/- 3 mmHg; and DeltaHR, -50 +/- 4 beats/min). The antihypertensive response was unaffected by the infusion of a pegylated catalase or by the inhibition of catalase with 3-aminotriazole, inhibition of glutathione peroxidase with buthionine sulfoximine, inhibition of heme oxygenase with tin mesoporphyrin, or inhibition of large-conductance Ca(2+)-activated potassium channels with iberiotoxin. However, the antihypertensive response was significantly (P < 0.01) blunted by 48% by the activation of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channels with cromakalim during maintenance of blood pressure with norepinephrine and by 31% by the blockade of these channels with glibenclamide, by 40% by the blockade of nitric oxide synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME), and by 40% by the blockade of ganglionic autonomic neurotransmission with hexamethonium. L-NAME and hexamethonium were additive, but glibenclamide and hexamethonium were less than additive. The central administration of Tempol was ineffective. The acute antihypertensive action of Tempol depends on the independent effects of potentiation of nitric oxide and inhibition of the peripheral SNS that involves the activation of K(ATP) channels.

Abstract-Angiotensin-converting enzyme inhibitors (ACEIs) decrease the glomerular filtration rate and renal blood flow in the clipped kidneys of early 2-kidney, 1-clip Goldblatt hypertensive rats, but the consequences for oxygenation are unclear. We investigated the hypothesis that angiotensin II type 1 or angiotensin II type 2 receptors or NO synthase mediate renal oxygenation responses to ACEI. Three weeks after left renal artery clipping, kidney function, oxygen (O 2 ) use, renal blood flow, renal cortical blood flow, and renal cortical oxygen tension (PO 2 ) were measured after acute administration of an ACEI (enalaprilat) and after acute administration of ACEI following acute administration of an angiotensin II type 1 or angiotensin II type 2 receptor blocker (candesartan or PD-123,319) or an NO synthase blocker (N G -nitro-L-arginine methyl ester with control of renal perfusion pressure) and compared with mechanical reduction in renal perfusion pressure to the levels after ACEI. The basal renal cortical PO 2 of clipped kidneys was significantly lower than contralateral kidneys (35Ϯ1 versus 51Ϯ1 mm Hg; nϭ40 each). ACEI lowered renal venous PO 2 , cortical PO 2 , renal blood flow, glomerular filtration rate, and cortical blood flow and increased the renal vascular resistance in the clipped kidney, whereas mechanical reduction in renal perfusion pressure was ineffective. 319 and N G -nitro-L-arginine methyl ester, but not candesartan, reduced the PO 2 of clipped kidneys and blocked the fall in PO 2 with acute ACEI administration. In conclusion, oxygen availability in the clipped kidney is maintained by angiotensin II generation, angiotensin II type 2 receptors, and NO synthase. This discloses a novel mechanism whereby angiotensin can prevent hypoxia in a kidney challenged with a reduced perfusion pressure. A reduced renal perfusion pressure (RPP) after clipping of a renal artery in the early (2-to 4-week) 2-kidney, 1-clip (2K,1C) rat model of Goldblatt hypertension increases angiotensin II (Ang II) concentrations in both kidneys 1 and causes Ang II-dependent hypertension. 2-4 A reduced renal tissue oxygen tension (PO 2 ) developing during prolonged infusion of Ang II 5-7 has been ascribed to reactive oxygen species and functional NO deficiency. Prolonged administration of the antioxidant drug Tempol, but not the angiotensin receptor blocker (ARB) candesartan, restores renal tissue PO 2 in a rat model of early 2K,1C hypertension. 5 This may be important, because renal hypoxia, and episodes of renal ischemia, may contribute to hypertension 8 and progressive kidney disease. 9 On the other hand, acute infusions of Ang II into rats increase renal NO generation and increase the dependency of renal blood flow (RBF) on NO. 10,11 Moreover, studies in the early 2K,1C rat model [12][13][14] have shown that the acute administration of an angiotensin-converting enzyme inhibitor (ACEI), or nonselective angiotensin receptor blockade with saralasin, reduces the RBF, and thereby the renal oxygen (O 2 ) delivery, and the glom...